The present invention relates to a flow cytometry method and apparatus for distinguishing and/or characterizing cells or particles on the basis of measured fluorescence lifetimes. More particularly, the present invention concerns a flow cytometry method and apparatus for distinguishing and characterizing a particle or cell which has been labeled or associated with one or more fluorophores having lifetimes which are modified due to one or more characteristics or properties of a cell or particle with which the fluorophore is associated, wherein the lifetime measurments are independent of the intensity of the detected fluorescence emission.
Research involving the study and analysis of cells, generally known as cytology, employs a variety of analytical techniques for identifying and enumerating the subpopulations of cells in a specimen under study. For example, cytological materials may be examined to detect the presence of cancerous or malignant cells, or characteristics of the cells within a specimen. For purposes of analysis, the cells may be labeled with a variety of fluorescent materials, conventionally known as fluorophores or fluorescent probes, which have an identified affinity for cells or cell components which are of interest to an analysis. The fluorophores will emit a particular fluorescence radiation when stimulated by light at a wavelength corresponding to the excitation wavelength of the fluorophore. The wavelength and/or intensity of the emitted light has been used to analyze a subpopulation of cells, wherein different fluorophores can be used to distinguish subpopulations of fluorophore-labeled cells.
The study of collections of multiple cells using fluorescent spectroscopy has obvious limitations. For example, an accurate determination of the number of cells in a subpopulation having a given characteristic cannot be made and, more importantly, the subpopulations cannot be separated for further analysis. In order to permit the measurement and analysis of a population of cells (or any biological particle such as isolated nuclei, chromosome preparations or neurobiological organisms) on an individual basis, fluorescence flow cytometry has been employed.
Fluorescence flow cytometry, which involves the intensity and/or wavelength measurement of fluorescence emissions from individual cells labeled with a fluorophore while the cells are flowing in a liquid or gaseous medium past an observation point, permits analysis of individual cells as well as sorting of the cells based upon that analysis. A description of examples of fluorescence flow cytometry appears in "Practical Flow Cytometry", 2d. ed., H. Shapiro (1988), Liss & Co, the entire contents of which are herein incorporated by reference.
A conventional flow cytometer has a sample handling and delivery system which collects the cell population into a stream of individual cells which is directed one cell at a time past the observation point of the flow cytometer. When a liquid medium is used, the stream containing the samples is sheathed by a surrounding fluid stream to insure that only single cells pass the observation point.
A conventional flow cytometer also has a parameter detection system which can include a focused light source, typically a laser, that directs a narrow beam of light at a predetermined wavelength to the observation point where an individual fluorophore-labeled cell passing through the point may be illuminated, resulting in a fluorescence emission from the fluorophore label. The parameter detection system also includes collection optics and optical transducers, such as photomultipliers and detectors that receive the fluorescence emission at the observation point and convert it to electrical signals which are representative of the intensity and/or wavelength of the emitted light.
Where the cells have been tagged with a fluorophore having an affinity for a particular characteristic or composition of the cell, as well as being excited at the wavelength of light emitted by the laser, the light emission intensity and/or wavelength of the fluorophore bound to each cell at the observation point may be detected for purposes of analysis.
When the fluorophore bound to a cell is excited by light at the fluorophore's absorption wavelength, the fluorophore's electrons can absorb energy such that the energy level of an electron is raised from the ground state to an excited state. The excited electron then emits a photon as it returns to the ground state, with the photon having a characteristic wavelength and intensity.
In FIG. 1 is shown an advanced streak camera flow cytometry apparatus. The pulsed output from the light source 1 passes through an optics subsystem 2 where it is shaped and focused into the continuous flow of cells or particles. The cells are in a suspension from sample source 4 and are aligned within sample chamber 5 by the laminar flow of a fluid sheath from a solution source 6. When the laser light illuminates an individual cell at the observation point in a flow chamber 3, a fluorescence is emitted as pulsed fluorescence radiation 7. This pulsed radiation is captured by an optics subsystem 8 and focused onto the streak camera detector 9. The output of the detector is a voltage that is applied by line 16 to a signal processing system 10 where it is used for cell population analysis based on intensity and/or wavelength of the emitted fluorescence. The signal processing is fast enough to control the division of the observed cells into subpopulation collections by a conventional cell sorter 11.
Thus, measurements of the intensity and/or wavelength of emitted fluorescence over a period of time may be used as the basis for discrimination and sorting of the cells or particles in the population under study, and the intensity of emitted light may be measured by photomultipliers.
Several conventional methods for distinguishing cells by detecting the emitted fluorescence light at the observation point of a flow cytometer system are known. The advanced streak camera system of FIG. 1 measures for each cell the change in the intensity of transient emitted light over a period of time as the cell passed through the laser beam. Based upon such measurements, the attenuation time of the emitted light, the rise time of the emitted light and the orientation relaxation time may be detected and used as a basis for cell discrimination. Referring again to FIG. 1, the attenuation time of the emitted light (i.e. the fluorescence lifetime, representing the average amount of time a molecule remains in the excited state prior to returning to a ground state) is determined based on the intensity of the fluorescence emission, using the output of streak camera 9 on a cell-by-cell basis. The output from camera 9 and a peak threshold value 12a are input to comparator 13, and, when a decreasing light emission signal reaches the peak threshold value, a counter 15 is started and continues to count until the light emission signal reaches an attenuation threshold value 14 (typically 1/e or 63% of peak) when the count is terminated. The processor 10 uses the attenuation time count for each cell to perform an analysis of the sample cell population and even control cell sorting with sorter 11.
However, this streak camera approach encounters significant difficulties due to the need for highly accurate and expensive counters. Also, this approach is dependent on the intensity of the emitted light, which will require the peak threshold to be varied. Moreover, if variable signal attenuation is present, due to the dependence of the count on emitted light intensity, the measuring of attenuation time may not be consistent. Furthermore, the use of a synchro-scan streak camera has the disadvantage of high cost, high complexity and limited sensitivity, owing to its extremely small sensitive area.
In a similar intensity-based system disclosed in U.S. Pat. No. 4,778,539, Yamashita et al, issued Oct. 18, 1988, individual cells may be distinguished by measuring light at an observation point of a flow cytometer system. There, the change in the intensity of transient emitted light over a period of time, following excitation by short pulses of laser light, may be measured and used to detect the attenuation time of the emitted light, the rise time of the emitted light and the orientation relaxation time. These parameters may be used as a basis for cell discrimination. However, such measurements are intensity-based and are performed cell-by-cell, and do not allow for rapid and/or simultaneous scanning of a population of cells, based on lifetime measurements.
A suggestion that phase based measurement of fluorescence lifetimes may be employed in flow cytometric systems appears in Cytometry, Supplement 2, p. 91 (1988), Steincamp et al. However, there is no specific disclosure of the structure or method of operation of a system which can perform the identified function.
Thus far, practitioners have been unsuccessful in measuring fluorescence lifetimes with phase-based techniques and in using such measurements to identify the existence of particular analytes without complicated processing of the detected data signals. Indeed, where such signal processing is used to determine the lifetime of radiated cells within a flow cytometer, the performance of a selective sorting of such cells has been difficult since the processing may not be sufficiently rapid to enable sorting of the cells as they flow through the cytometer observation point.
In a system not previously used for flow cytometry, phase-modulation fluorometry and phase-sensitive fluorescence spectroscopy (PSFS) provide a means by which fluorescence lifetimes of one or a few fluorophores in homogeneous solutions are measured for the study of specific fluorophore lifetimes. One approach, described in PRINCIPLES OF FLUORESCENCE SPECTROSCOPY, J. R. Lakowicz, Plenum Press (1983), discloses a technique in which a sample containing only one or a few fluorophores is excited with light having a time-dependent intensity and a detection is made of the resulting time-dependent emission. Because the emission is demodulated and phase shifted to an extent determined by the fluorescence lifetime of the species, the fluorescence lifetime (.tau.) can be calculated from the phase shift O of the species: ##EQU1##
or from a demodulation factor m: ##EQU2## where m is a demodulation factor, .omega. is the angular modulation frequency and O is the phase shift of the species. Phase sensitive detection results from a comparison of the detected emission with an internal, electronic reference signal of the same frequency.
In a second approach, described in "Phase-Sensitive Fluorescence Spectroscopy: A New Method To Resolve Fluorescence Lifetimes Or Emission Spectra Of Components In A Mixture Of Fluorophores" Lakowicz et al, Journal of Biochemical and Biophysical Methods; vol. 5, 1981, pp. 19-35, the time-dependent fluorescence photodetector output signal is multiplied by a periodic square-wave signal which has the same modulation frequency as the fluorescence signal, and is then integrated. A time-independent dc signal is thereby produced that is proportional to the cosine of the difference between the phase angles of the square-wave and the fluorescence signal, and proportional to both signal amplitudes.
The two approaches described above are related to periodically modulated fluorescence excitation light. Consequently, continuous output signals are generated in both techniques. Therefore, these standard techniques would not have been considered to be used to measure fluorescence lifetimes of single cells passing the observation region in a flow cytometer. Moreover, as already mentioned, the output signal in the second technique depends not only on the fluorescence lifetime but also on the fluorescence signal intensity. As a matter of experience, the fluorescence signal intensity observed in a flow cytometer varies significantly from cell to cell, and therefore phase-modulation fluorometry and PSFS would not have been considered to be useful for flow cytometric measurements. Also, known phase modulation fluorometry methods use slowly responding circuits which would be expected to prevent cell-to-cell measurements.